Proteoglycan compositions for treating arthritic inflammatory conditions

ABSTRACT

Compositions with synergistic anti-inflammatory effects in inflammatory diseases resulting from activation and consequent degranulation of mast cells and followed by secretion of inflammatory biomolecules from the activated mast cells, composed of a heavily sulfated, non-bovine proteoglycan such as shark cartilage chondroitin sulfate C, and one or more of a hexosamine sulfate such as D-glucosamine sulfate, a flavone such as quercetin, an unrefined olive kernel extract that increases absorption of these compositions in various routes of administration, S-adenosylmethionine, a histamine-1 receptor antagonist, a histamine-3 receptor agonist, an antagonist of the actions of CRH, caffeine, and a polyamine.

This application is a continuation/divisional of co-pendingPCT/US02/00476, filed Jan. 3, 2002, which is a continuation ofco-pending U.S. Ser. No. 09/771,669, filed Jan. 30, 2001.

BACKGROUND OF THE INVENTION

The invention is generally related to the treatment of inflammatoryconditions. More specifically, the invention is related to compositionscontaining inhibitors of mast cell activation and secretion such as aproteoglycan that are designed to be used as dietary supplements oradjuvants to conventional approved medications for the relief ofinflammatory conditions.

There have been a number of mostly anecdotal reports that theproteoglycan chondroitin sulfate, as well as glucosamine sulfate, aproduct of the intestinal breakdown of proteoglycans, may be helpful inrelieving the pain of osteoarthritis:—Shute N. Aching for an arthritiscure. US News and World Report, Feb. 10, 1997.—Cowley G. The arthritiscure? Newsweek, Feb. 17, 1997; Foreman J., People, and their pets, toutarthritis remedy. The Boston Globe, Apr. 7, 1997; Tye L. Treatment gainsscientific attention. The Boston Globe, Sep. 25, 2000.

A recent meta-analysis showed potential therapeutic benefit ofchondroitin sulfate and/or glucosamine in osteoarthritis [McAlindon etal. J Am Med Assn. 283:1469 (2000)], while a double-blind clinical trialwith glucosamine showed definite benefits in osteoarthritis with respectto both pain and radiographic joint appearance [Reginster et al., Lancet337:252 (2001)]. However, less than 5% of the chondroitin sulfate incommercially available preparations is absorbed orally, because the sizeof the molecule and the degree of sulfation impede its absorption fromthe gastrointestinal tract. Furthermore, such commercial preparationsuse chondroitin sulfate obtained from cow trachea, with the possibledanger of contracting spongiform encephalopathy or “mad cow disease”. Infact, the European Union has banned even cosmetics that containbovine-derived products.

Theoharides et al. British Journal of Pharmacology 131:1039 (2000)indicated for the first time how proteoglycans such as chondroitinsulfate may work. The paper reported that chondroitin sulfate and, to alesser degree, glucosamine sulfate, inhibit activation of mast cellsthat are known to trigger allergy and asthma. This discovery is thebasis for Theoharides, U.S. patent application Ser. No. 09/056,707,filed Apr. 8, 1998 and Ser. No. 09/773,576, filed Feb. 2, 2001.

Mast cells are also now recognized as important causative intermediaryin many painflul inflammatory conditions[Galli, N Eng J Med. 328:257(1993); Theoharides, Int J Tissue Reactions 18:1 (1996)], such asinterstitial cystitis and irritable bowel syndrome [Theoharides, Ann NYAcad, Sci. 840:619 (1998)], as well as in migraines and possiblymultiple sclerosis [Theoharides, Persp Biol Med. 26:672 (1983);Theoharides, Life Sci 46:607 (1996)]. In fact, glucosamine was recentlyconsidered to be prophylactic for migraines [Russell, Med Hypoth 55:195(2000)].

Mast cells are increasingly implicated in conditions involving inflamedjoints, such as in osteoarthritis and rheumatoid arthritis, throughactivation of local mast cells by, for example, neuropeptides, such asSubstance P. Additional indirect evidence also supports the involvementof mast cells in bone resorption: (a) systemic mastocytosis isinvariably associated with osteoporosis; (b) inhibition of mast cellmediator release reversed lytic bone changes; (c) depletion of mastcells inhibited bone resorption in organ culture; (d) human synovialmast cells were shown to secrete in response to allergic andnon-immunologic stimuli; (e) human mast cells release the cytokine IL-6and (f) IL-6 has been definitively linked to bone resorption andosteoporosis.

It was recently shown that chondroitin sulfate's ability to inhibit theactivation of mast cells compliments the inhibitory effects on mast cellactivation of another class of naturally occurring compounds, theflavonoids [Middleton et al. Pharm Rev 52:1 (2000)]. Certain plantflavones (in citrus fruit pulp, seeds, sea weed) are now recognized asanti-allergic, anti-inflammatory, anti-oxidant and cytoprotective withpossible anti-cancer properties. Only some flavonoids that belong to thesubclass of flavones, e.g., quercetin, inhibit mast cell activation.

Quercetin inhibits secretion from human activated mast cells [Kimata etal. Allergy 30:501(2000)], and has also been used effectively for thetreatment of chronic prostatitis [Shoskes et al., Urology 54:960(1999)]. However, other flavonoids may have opposite effects. Use of theterm “bioflavonoids” or “citrus flavonoids” in certain commercialproducts, therefore, provides little information, and may includemolecules that have detrimental effects; for example, soy containsisoflavones that have estrogen-like activity that worsens inflammatoryconditions.

Copending U.S. patent application Ser. No. 09/056,707, filed Apr. 8,1998, and divisional Ser. No. 09/773,576 claim the oral use ofproteoglycans, without and with flavonoids, for the treatment of mastcell activation-induced diseases. Absorption of these compositions fromthe gastrointestinal tract and synergism with other treatment modalitieswere not addressed in these applications.

Applicant has described the use of antagonists of the action ofCorticotropin Releasing Hormone (also known as Corticotropin ReleasingFactor) in inhibiting myocardial mast cell activation in myocardialischemia (copending U.S. patent application Ser. No. 08/858,136, filedMay 18, 1997), in treating stress-induced skin disease (U.S. Pat. No.6,020,305) and stress-induced migraine headaches (U.S. Pat. No.5,855,884), the contents of which are incorporated herein by reference.The synergistic effects of the compositions of the present inventionthat include antagonists of the actions of Corticotropin ReleasingHormone (“CRH”) on mast cells were not recognized at the time of theprevious studies. The word “antagonists” in connection with CRH isintended herein to include any molecule that prevents the actions of CRHon target cells, and includes, but is not limited to, anti-CRHneutralizing antibodies or binding proteins, or molecules preventing therelease of CRH at local sites (see below for details).

Applicant has also described a method for treating patients with mastcell derived molecules-induced interstitial cystitis with histamine-1receptor antagonists (U.S. Pat. No. 5,994,357). Treatment of mast cellmolecules-induced migraines with histamine-1 receptor antagonists is thesubject of Theoharides U.S. Pat. No. 5,855,884. Histamine-3 receptoragonists as pharmaceutical agents in mast cell-involved diseases aredescribed in Theoharides U.S. Pat. No. 5,831,259. The contents of thesethree patents are incorporated herein by reference. At the time of thisinvention the synergistic effects of the present compositions with suchantagonists had not yet been recognized.

An important need therefore exists for compositions for administrationto human patients being treated for mast cell-induced inflammatorydiseases by various modalities, that are synergistic in that they havestronger effects than the sum of the effects of the individualcomponents, and also synergistic with conventional clinical treatmentsof inflammatory conditions. “Synergistic” is also intended to mean:“coordinated or correlated action by two or more structures or drugs”[Stedman's Medical Dictionary, 23rd edition, Williams & Wilkins,Baltimore, 1976]. An important need also exists for formulations thatincrease the absorption from the gastrointestinal tract, nasal passagesand skin surface of the compositions of the invention. Such formulationshave been discovered, and are described below.

SUMMARY OF THE INVENTION

The invention comprises compositions for human use containing a sulfatedproteoglycan and an unrefined olive kernel (seed) oil, and one or moreactive ingredients selected from the group consisting of a sulfatedhexosamine, a flavonoid compound, S-adenosylmethionine (“SAM”),histamine-1 receptor antagonists, histamine-3 receptor agonists,antagonists of the actions of CRH, caffeine, folic acid, rutin,polyunsaturated fatty acids, and polyamines, together with appropriateexcipients and carriers, said compositions having improved absorptionfrom the gastrointestinal tract, skin surface, and nasal and pulmonarysurfaces, and anti-inflammatory effects synergistic with each other andsynergistic with available conventional clinical treatment modalities.

In one embodiment, the sulfated glucosamine is D-glucosamine sulfate,the proteoglycan is non-bovine chondroitin sulfate, and the flavone isquercetin.

In an other embodiment, compositions may also contain antagonists of theeffects of CRH on mast cells or other target cells of the myocardium,gastric mucosa, urinary bladder, skin, meningeal membranes, andblood-brain barrier.

In still another embodiment, the present compositions are used againstsuperficial vasodilator flush syndromes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

It has been discovered that a combination of a sulfated proteoglycan, asulfated D-hexoseamine and a flavone in a unique, unrefined olive kernelextract, with optional CRH antagonists, histamine-1 receptorantagonists, histamine-3 receptor agonists, polyamines and caffeine hassynergistic anti-inflammatory effects when used as a dietary supplement,a topical product or an aerosol for nasal or pulmonary adminstration,without or with a conventional clinical treatment for inflammatorydiseases. Such inflammatory diseases result from the activation,degranulation and consequent secretion of inflammatory biochemicals frommast cells, and the resultant inflammatory diseases include the groupconsisting of: allergic inflammation, arthritis (to includeosteoarthritis and rheumatoid arthritis), cancer, fibromyalgia,inflammatory bowel disease, interstitial cystitis, irritable bowelsyndrome, migraines, angina, chronic prostatitis, eczema, multiplesclerosis, psoriasis, sun burn, periodontal disease of the gums,superficial vasodilator (flush} syndromes and hormonally-dependentcancers.

In a highly preferred embodiment, the sulfated proteoglycan isnon-bovine chondroitin sulfate, preferably from shark cartilage, whichblocks mast cell activation, degranulation and consequent secretion ofinflammatory biochemicals from the mast cells. Other natural sulfatedproteoglycans suitable for practicing this invention include keratansulfate, dermatan sulfate and hyaluronic acid sodium salt (sodiumhyaluronate). A preferred biological source of the chondroitin sulfateis shark cartilage which is more-highly sulfated than the commoncommercial chondroitin sulfate isolated from cow trachea; the sharkcartilage source also avoids the potential dangers associated withbovine sources.

The highly preferred flavone is quercetin which inhibits secretion ofinflammatory molecules from mast cells by affecting moesin, a unique 78kDa mast cell protein [Theoharides et al. J Pharm Exp Therap 294:810(2000)]. In addition to quercetin, other flavones suitable in carryingout the invention include myricetin, genistein, kaempferol and thequercetin glycoside rutin. A highly preferred source of quercetin andits glycoside is the Saphona plant.

The olive kernel (pit) extract component of the inventive compositionsis preferably an unrefined (first pressing, filtered, oleic acid-relatedacidity <1%, water content <5%) oil produced, for one source, on theisland of Crete in Greece. This kernel oil is especially prepared by themaker by a process consisting essentially of: (1) washing the kernelmass that remains after the compression of the oil from the olive fleshwith water (Sansa); (2) drying the washed kernels in a stream of hot airat about 80 degrees C. to reduce the humidity to about 1%; (3)extracting the dried kernels with hexane and steam; (4) cooling thehexane extract, microfiltering the extract (5 micron pore size) toremove particulate matter; (5) heating the hexane extract at about 40degrees C. degrees while percolating helium (to avoid oxidation) throughthe fluid to evaporate the hexane (final <0.5%), which process reducesthe water content to <1% and the acidity (as oleic acid) to <3%; and (6)storing the extract in sealed containers. This olive kernel extractsurprisingly has the unique property of increasing absorption of theother components of the anti-inflammatory compositions through theintestinal mucosa and skin, and also adds its own content of importantanti-oxidants [Bosku, World Rev Nutr Diet, 87:56 (2000)], such as omegafatty acids (e.g. eicosapentanoic acid) and alpha tocopherol. Althoughnot claimed herein, it has been claimed that kernel olive extract hascytoprotective, longevity-producing effects [Trichopoulou et al. Am JClin Nutr 61:1346S (1995); Trichopoulou et al, Cancer Epid BiomarkerPrevention 9:869 (2000)]. The polyphenols in such olive oil also haveanti-inflammatory effects in, for example, arthritis [Martinez-Dominguezet al., Inflamm. Res. 50:102 (2001)]. A preferred source of theunrefined olive kernel extract of the invention is: E.B.E.K., Inc.,Commercial, Industrial Enterprises of Crete, 118 Ethnikis Antistasecos,Heraklion, Crete, 71306, Greece.

Supplementation of the compositions described above with the methylationreagent S-adenosylmethionine (“SAM”) adds antioxidant, anti-inflammatoryand cytoprotective properties, particularly in inflammatory jointdiseases. Addition of SAM also accelerates metabolism of homocysteine,which amino acid has been implicated in coronary disease, to cysteine,which is harmless. Folic acid may be added to certain of the presentformulations for similar reasons.

Another supplement to the basic compositions of the invention is ahistamine-1 receptor antagonist, such as diphenhydramine, hydroxyzine,azelastine, azatadine and cyproheptadine. Other histamine-1 receptorantagonists are described in Table 25-1 in Goodman and Gilman's ThePharmaceutical Basis of Therapeutics, 9^(th) ed., New York, 1996.Histamine-3 receptor agonists are described in the Theoharides patentslisted above.

Inhibitors of mast cell activation and secretion may be used in thetreatment of inflammatory processes such as superficial vasodilatorsyndrome, e.g., menopausal-associated flush, monosodiumglutamate-associated flush, carcinoid flush and niacin-associated flush.

Sources of CRH antagonists include, in addition to the Theoharidespatents listed in the Background section above: Neurocrine Biochem.Inc.'s D-Phe 12 Nle Ala32,21,38hCRH(12-41)NH2, cat no. 1P-36-41; Pfizernon-peptide CP-154,526-1; Sigma Chem., St. Louis anti-CRH polyclonalantiserum; and Pfizer, N.Y. patents and applications: U.S. Pat. Nos.6,211,195, 5,795,905, PCT/IB95/00573, PCT/IB95/00439, U.S. Ser. Nos.08/448,539, 08/481,413, 09/735,841, and in Owens et al. Pharm. Rev.43:425 (1991).

The preferred concentration range of the proteoglycan, hexosaminesulfate and flavone components of the oral formulations are 10-3,000 mgper tablet or capsule. The preferred concentration range for SAM is3-1,000 mg per capsule or tablet. Generally, where present, the amountsof the unrefined kernel oil are at least three times those of the otheractive ingredients, preferably 900-1200 mg. The number of capsules ortablets to be taken per day is determined by the nature and severity ofthe medical condition, and is readily determinable by the patient'shealth provider. Other representative formulations are described in theexamples below.

The compositions of the invention may be formulated in any standardmeans of introducing pharmaceuticals into a patient, e.g., by means oftablets or capsules. The compositions of the invention include ointmentsand creams for skin conditions, mouth washes and toothpaste forperiodontal diseases, and solutions for nasal aerosols. Standardexcipients and carriers for the active ingredients of the inventivecompositions are described in Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa. Fragrances and flavorings may also be added.

Although not bound by any particular mechanism of action of thecomponents of the claimed compositions, the inventor contemplates thatthe proteoglycan inhibits the activation and degranulation of therelevant mast cells, while the flavone inhibits the secretion ofinflammatory biomolecules from these mast cells. “Activation” and“degranulation” of mast cells are defined herein as is standard and wellknown in this art, that is, to mean secretion from the activated mastcell of any type of molecule(s) that alone or in combination triggersinflammatory processes.

EXAMPLES Example 1

Table 1 compares chondroitin sulfate-containing commercial products tothe present compositions.

TABLE 1 Comparison of Chondroitin Sulfate-Containing Products to PresentInvention Most Available Product Compositions Present Invention MainMixture of chondroitins Non-bovine chondroitin ingredient sulfate,preferably the C type Source Cow trachea Shark cartilage Amount per100-300 10-3000 mg capsule or tablet Degree of Low, if any Highsulfation Absorption <5% >15% from g.i. tract Target Unknown Mast cells,inflammatory cells Other Vitamins, fish oils Flavones, unrefined kernelingredients (some preparations) olive oil, SAM, histamine-1 receptorantagonists, histamine-3 receptor agonists, CRH antagonists, polyamines,caffeine, folic acid Advantages None known Anti-allergic, anti-inflammatory, anti-oxidant, cytoprotective Adverse effects Risk of madcow disease, None known spongiform encephalopathy, stomach upset,allergy to fish products Relevant Osteoarthritis Allergic inflammationconditions angina, asthma coronary artery disease, arthritis(osteoarthritis or rheumatoid arthritis), chronic prostatitis, eczema,fibromyalgia, interstitial cystitis, irritable bowel syndrome,inflammatory bowel disease, migraines, multiple sclerosis, psoriasis,periodontal disease, flush syndrome, cancer (includinghormonally-dependent forms). Scientific None found Theoharides et al. BrJ publications Pharm 131:1039 (2000) Middleton et al. Pharm Rev 52:673(2000)

In all examples, chondroitin sulfate is to assumed to be of a non-bovinevariety.

Example 2 Composition for Protecting Against Inflammatory Diseases

Two capsules to be taken orally 2-3 times daily, at least one hourbefore meals

Ingredients, per capsule, mg: * Chondroitin sulfate 150-300 *D-Glucosamine sulfate 150-300 * Quercetin 150-300 * Unrefined olivekernel extract  900-1200

Example 3 Composition for Protecting Against Arthritis

Ingredients per capsule, mg: *D-Glucosamine sulfate 150-300 *Chondroitinsulfate 150-300 *Sodium hyaluronate 100-200 *Quercetin 150-300*Unrefined olive kernel extract  900-1200

Example 4 Topical Composition for Protecting Against Arthritis

Skin ointment or cream. Apply three times per day to affected areas.Ingredients % by weight *D-glucosamine sulfate 5 *Chondroitin sulfate 5*Sodium hyaluronate 5 *Bitter willow bark extract 5 *Quercetin 3*Unrefined olive kernel extract 15 

Example 5 Composition for Protecting Against Cardiovascular Disease

Two capsules to be taken orally 2-3 times per day, in mg: *Chondroitinsulfate  50 *Kaempferol 100 *S-adenosylmethionine  50 *Niacin 100*Unrefined olive kernel extract  900-1200 *Bitter willow bark extract,5% by weight

Example 6 Composition for Protecting Against Periodontal Disease

Mouthwash: *Chondroitin sulfate 0.4 M *Quercetin 0.4 M *In a standardmouthwash vehicle

Example 7 Toothpaste Composition

Toothpaste, mg %: *Chondroitin sulfate 5 *Quercetin 3 *Optionally,D-glucosamine sulfate 5 *In a standard toothpaste vehicle

Example 8 Sunscreen Composition

Ingredients mg % *Chondroitin sulfate 5 *D-glucosamine sulfate 5*Quercetin 3 *Sun screen (e.g., TiO₂) 5

Example 9 Composition for Protecting Against Migraine Headaches

Ingredients, mg: *Chondroitin sulfate  50 *Quercetin 100 *Azatadine  4 *Optionally, a CRH-receptor antagonist

Example 10 Composition for Protecting Against Relapsing MultipleSclerosis

Ingredients, mg: *Chondroitin sulfate  50 *Quercetin 400 *Hydroxyzine 50 *Optionally, interferon-beta

Example 11 Composition for Protecting Against Cystitis and Prostatitis

Ingredients, mg: *D-glucosamine sulfate 50 *Chondroitin sulfate 100-300*Sodium hyaluronate 200 *Quercetin 100-400 *Unrefined olive kernelextract  900-1200

Example 12 Composition for Protecting Against “Flush”

Ingredients, per capsule: *Chondroitin sulfate  50 mg *Quercetin 150 mg*Bitter willow bark extract 5% by weight *Optionally, cyproheptadine or 4 mg  azatadine

Example 13 Cream Composition for Protecting Against Skin Allergy

Ingredients: % by weight *Aloe vera 5 *Non-bovine chondroitin sulfate 5*Myricetin 5 *Alpha-tocopherol  l 5 *Unrefined olive kernel extract 15 *Optionally, azelastine or hydroxyzine 5

Example 14 Composition for Protecting Against Allergy and AllergicAsthma

Ingredients, mg Myricetin 500 Chondroitin sulfate 200 Optionally,azelastine or hydroxyzine

Example 15 Composition for Protecting Against Hormonally-DependentCancers

Ingredients, mg Quercetin 150 Genestein  50 Optionally, tomoxifen orraloxifen  10

Example 16 Composition for Protecting Against Allergic Conjunctivitis

Ingredients: *Quercetin 0.05% *Chondroitin sulfate  2.0% *Optionally,azelastine 0.05%

I claim:
 1. A composition with synergistic anti-inflammatory propertiesin arthritic conditions induced by the activation of mast cells,consequent degranulation of said cells and subsequent secretion ofinflammatory biomolecules, comprising: shark cartilage chondroitinsulfate, 150 mg; Saphona plant quercetin, 150 mg; shellfish chitinD-glucosamine sulfate; unrefined, microfiltered olive kernel (pit)extract of acidity less than about 3% and water less than about 5%, 55%;and, as suspending agents, 40 mg beeswax and 20 mg lecithin, in soft gelcapsules made from pig gelatin.